The objective of this investigation was study the physiologic and proteomic response to hydric deficit in vitro induced by PEG-8000, in calli of four cultivars of onion, 'Sivan' and 'Americana' (red), 'Ica' and 'Ram' (yellow). For all the cultivars without acclimatization treatment (without PEG) the survival percentage diminished in relation to increasing of concentration of PEG, whereas with acclimatization (10% of PEG-8000) the survival percentage increased in 'Ica' and 'Sivan', and it diminished in 'Americana' and 'Ram' al lowest concentrations of PEG. 'Ica' and 'Americana' presented the highest percentage of survival at 40% of PEG, 'Sivan' increased to 47%, and 'Ram' shown its lowest value (22.8%). The value of DHL50 in calli with-PEG, increased significatively in 'Ica' (11.38% PEG w/v) and 'Sivan' (10.5% de PEG w/v).With-PEG, the content of carbohydrates diminished in 'Americana'. In 'Sivan' and 'Ica' the carbohydrates increased and in 'Ram' the increasing was twofold. The proline level increased in 'Ica', 'Sivan' and 'Americana'; but it diminished in 'Ram'. The concentration of soluble protein increased in all cultivars. The preliminary proteomic analysis shows proteins related with the growth cellular and enzymes related with the activity Ascorbate peroxidase and Superoxide dismutase in 'Ica' and 'Sivan' with capacity to acclimatization, indicating a possible role of these proteins in the tolerance to hydric deficit.